What is best method for obtaining Fetal hemoglobin from red blood cells?
Do you think it is mass spectrometry? I am open for any suggestions.
to extract it, you would probably do LC.
Mass spectrometry can measure it, but it will consume the sample. So, whatever you analyzed will be gone.
Dear Sonja, LC stands for Liquid chromatography? or it means HPLC? Do you know maybe some cheaper technic? Thank you.
Yes, LC stands for Liquid Chromatography and usually refers to the High Pressure Liquid Chromatography version. I suppose if you do not have an HPLC instrument, you could try to use C18 spin columns (either make them yourself or buy them from Pierce) and try to achieve separation that way.
Dilute your blood sample in 5% acetic acid at least 1:10, add 100 ul of that to your spin column and centrifuge. Then start using increasing amounts of acetontirile (10, 20, 30, 40% etc.), centrifuge and capture the flowthrough. You might need to do this a couple of times to figure out what percentages are just right to separate your HbF, which should elute last.
Another alternative would be to fill an empty column with C18 material and let it just drip through. Gravity will take care of this. It just takes much longer than the spin column version. You will still need to apply a gradient over time.
Both of these approaches are cheaper than HPLC.
Do you think I can also purify hemolysate with anion exchange chromatography ? I mean, are you referring to that kind of LP?
If I use Alkali denaturation test, do you think I will destroy or damage Hb F?
Maybe, we should first clarify as to what your goal of this experiment is.
Most chromatographic procedure when done under denaturing conditions will not destroy your protein, but may denature it, so that it will not be functional anymore after your isolation.
I have done only analyses to quantify HbF vs HbA and HbB using LC-MS, where that did not matter. You were initially talking about MS, so I assumed you have a similar goal.
Yes, I understand. I will use HbF afterwords for further experiment. I need HbF more or less active, not deactivated. Is it possible to achieve that from hemolysate?
In that case, maybe this would be the best procedure to follow:
http://www.sciencedirect.com/science/article/pii/0022175980901908
I do not have access to the full publication though.
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